The Staudt laboratory has conducted RNA interference genetic screens for genes required for the proliferation and/or survival of human cell lines representing various subtypes of lymphoma and multiple myeloma. In diffuse large B cell lymphoma (DLBCL), previous work in the Staudt laboratory demonstrated that the anti-apoptotic NF-kB pathway is constitutively active in the activated B cell-like (ABC) subtypes of DLBCL but not the germinal center B cell-like (GCB) subtype of DLBCL, but the mechanisms underlying this abnormal signaling were enigmatic. The laboratory therefore conducted an RNAi screen in ABC and GCB DLBCL cell lines, searching for shRNAs that were selectively toxic for ABC DLBCL cells. This effort revealed that a signaling complex comprised of CARD11, MALT1, and BCL10 is required for the survival of ABC but not GCB DLBCL cell lines. In normal lymphocytes, this CARD11 complex engages the NF-kB pathway during antigen receptor signaling. The Staudt laboratory demonstrated that this signaling complex is responsible for the constitutive activation of the NF-kB pathway in ABC DLBCLs. In a recent RNA interference screen we identified several shRNAs targeting casein kinase 1A1 (CK1alpha) that were toxic for ABC but not GCB DLBCL cell lines. Since this phenotype resembled the toxicity profile of CARD11 shRNAs, we suspected that CK1alpha might be a new component of the CBM signaling pathway. Indeed, CK1alpha knockdown decreased IKK kinase activity and NF-kB target gene expression, and CK1alpha colocalized with CARD11 in ABC DLBCL cell lines. Fortuitously, Mike Lenardo's laboratory in NIAID separately identified CK1alpha as a binding partner of CARD11 in a mass spectrometry-based screen. Collaborative experiments revealed the essential role of CK1alpha in T cell receptor activation of the NF-kB pathway. Biochemically, CK1alpha was required for the recruitment of IKK to the CBM complex. Interestingly, T cells reconstituted with a CK1alpha mutant that was defective in kinase activity provided a greater NF-kB stimulus than did wild type CK1alpha, demonstrating that CK1alpha has both positive and negative influences on the CBM pathway. The negative effect of CK1alpha was due to its phosphorylation of CARD11 on serines in its "linker" domain, causing CARD11 to be destabilized. This negative feedback loop involving CK1alpha was analogous to the negative regulation of the CBM complex caused by inhibitory phosphorylation of BCL10 by IKK. CK1alpha emerged from this study as a new component of the CBM, demonstrating the power of unbiased RNA interference screens to uncover overlooked aspects of cellular signaling. A recent RNAi screen uncovered a crucial dependency of multiple myeloma cells on IRF4, a lymphoid-restricted transcriptional factor that is required for both lymphocyte activation and for plasmacytic differentiation. IRF4 knockdown by RNAi was toxic to 10 different myeloma cell lines representing many of the known genetic subtypes of this cancer. Of note, IRF4 is not translocated, amplified or mutated in most cases of multiple myeloma, and thus the dependency of myeloma cells on IRF4 exemplifies a new concept in cancer biology known as non-oncogene addiction. These results establish IRF4 as an important new therapeutic target in this lethal cancer. Most recently, our Achilles heel screens allowed us to define a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs with wild type CARD11. Such ABC DLBCLs die upon knockdown of BCR signaling components, including subunits of the B cell receptor itself. ABC DLBCLs have prominent clusters of the BCR in the plasma membrane, similar to antigen-stimulated normal B cells. Cancer gene resequencing revealed that over one fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. The most common mutations, present in 18% of ABC DLBCLs, involved a single tyrosine of the BCR signaling subunit, CD79B. These mutations affect the critical ITAM signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Importantly, the BCR pathway offers a wealth of targets that can be exploited therapeutically, including several protein kinases (SRC-family kinases, SYK, BTK, PKCbeta) as well as PI(3) kinase. Dasatinib, a clinically available kinase inhibitor that targets BTK and SRC-family kinases, kills ABC DLBCL cells by blocking their chronic active BCR signaling. An important new initiative in the past year was the construction of a new RNAi library that targets each gene with 12 shRNAs. This approach allows us to identify effective shRNAs that do not have appreciable off-target effects. This library targets all protein kinases, PI(3) kinase pathway component, and other regulatory proteins relevant to lymphoid malignancies. Using this new library we have embarked on an ambitious project to screen more than 50 cell line models of various lymphoid malignancies, searching for pathways that are selectively required for the proliferation and survival of particular cancer subtypes.